Pharmaceutical composition for preventing or treating brain damage due to neurological disease

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of brain damage caused by neurological diseases. The present invention provides a pharmaceutical composition for the prevention or treatment of brain damage comprising amlexanox.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for preventing or treating brain damage caused by neurological diseases. Background Technology

[0002] Zinc in the brain performs various physiological functions, including synaptic function, intracellular signal transduction, regulation of various genes (zinc finger transcription factors), autophagy, lysosomal function, and neurogenesis; therefore, zinc homeostasis is essential for maintaining normal brain function.

[0003] Some of the zinc in the brain is accumulated within synaptic vesicles by the zinc transporter zinc transporter 3 (ZnT3) and released from excitatory synaptic terminals during synaptic activity. Zinc is present in the terminal portions of glutamatergic axons throughout the mammalian central nervous system, including the hippocampus, cerebral cortex, and spinal cord (Danscher et al., 1985; Frederickson, 1989).

[0004] It is known that the secretion and accumulation of zinc contribute to neuronal death in several disease states, such as epilepsy (Frederickson et al., 1988; Suh et al., 2001), cerebral ischemia (Tonder et al., 1990; Koh et al., 1996), traumatic brain injury (Suh et al., 2000b), and hypoglycemia (Suh et al., 2004; Suh et al., 2007; Suh et al., 2008). In particular, zinc is known to contribute to the development of dementia by binding to amyloid beta peptides (Bush et al., 1994; Suh et al., 2000a; Cherny et al., 2001). This phenomenon is known to induce intracellular zinc deficiency, which exacerbates the symptoms of dementia.

[0005] Furthermore, zinc deficiency is prevalent in the elderly population, and since this can lead to autophagy and a decline in lysosome function, resulting in the accumulation of waste products and brain aging, normalizing zinc homeostasis is expected to help delay aging by normalizing lysosome function.

[0006] Since the underlying causes of vascular dementia also involve the aging of vascular endothelial cells and surrounding cells, reduced autophagy, accumulation of lipid waste products, resulting inflammatory responses, and disruption of the blood-brain barrier (BBB), it is thought that maintaining zinc homeostasis in these cells would be beneficial.

[0007] In the case of Alzheimer's dementia, the accumulation of Abeta and subsequent p-tau is associated with the decline in lysosomal function, so methods to induce an increase in zinc within the lysosome may be helpful as a treatment to reduce toxic proteins.

[0008] In addition to this, there is a possibility that a deficiency of synaptic zinc indirectly lowers BDNF / Trk signaling between neurons, which may contribute to the decline in cognitive function in dementia, so a method to improve cognitive function through the normalization of synaptic zinc may be helpful.

[0009] Most central nervous system cells are cells that have completed cell division, and therefore, in the case of humans, they must survive and maintain their functions for at least several decades. While divisible cells, such as blood cells, can process accumulated toxic waste products through cell division and apoptosis, nerve cells, which persist for a long time, are inevitably vulnerable to the accumulation of toxic waste. In other words, the central nervous system is the most severely affected when the function of lysosomes—the most critical organelle for waste removal—deteriorates.

[0010] The central nervous system contains not only neurons but also various types of "supportive cells," such as astrocytes, microglia, and oligodendrocytes. These cells are not only affected by toxic waste products but also play a role in absorbing and removing toxic waste released from neurons. However, excessive exposure to toxic waste products can contribute to neuronal damage through inflammatory responses. For example, it has recently been discovered that astrocytes transform into type 1 astrocytes that damage neurons, necessitating a clarification of their specific roles. Microglia also contribute to waste removal through their phagocytic function; however, when activated, they induce inflammation and secrete cytokines, which can also lead to neuronal damage. Furthermore, it is believed that vascular cells (endocytes, pericytes) and cells involved in CSF metabolism (ependymal cells, arachnoid granulation cells) undergo functional decline, thereby indirectly contributing to neuronal damage.

[0011] The mechanisms by which toxic waste products are generated in cells are diverse. For instance, toxic proteins or waste products are produced through various pathways, such as the generation of modified proteins due to genetic damage, improper protein folding caused by ER stress, impaired ubiquitin / proteasome function, metal metabolism abnormalities, and oxidative damage. However, if lysosome function is normal, it is possible to process these substances. The problem is that in long-lived cells like neurons, the effects of waste products accumulate over the long term, leading to a decline in lysosome function. In particular, most toxic proteins alkalize the pH of lysosomes, creating a vicious cycle. Therefore, developing methods to enhance lysosome function—specifically those effective when v-ATPAse, which acidifies lysosomes, is impaired—would not only reduce the accumulation of toxic waste products but also enable the development of fundamental treatments for brain damage caused by neurological diseases. Furthermore, given that p-tau accumulation occurs in vascular dementia, there is a possibility that enhancing lysosome function to remove it could be effective in treating vascular dementia.

[0012] To maintain the health of the central nervous system throughout the entire human lifespan, which is becoming increasingly longer, it is crucial to effectively and continuously remove waste products from nerve cells. However, there are currently no drugs or methods to effectively facilitate this process, and in particular, there are no drugs that effectively enhance the function of lysosomes, the primary organelles responsible for waste removal.

[0013] The inventors have discovered that zinc within lysosomes enhances the function of lysosomes in neurons and astrocytes. Therefore, appropriately regulating zinc metabolism in the nervous system may promote the removal of toxic waste products within the nervous system through the enhancement of lysosomal function. This strategy is expected to be useful for preventing and treating Alzheimer's and vascular dementia accompanied by lysosomal dysfunction (Fig. 1).

[0014] To date, no therapeutic agent for neurological diseases has been developed that targets the increased removal of waste products within neurons. Furthermore, strategies to achieve this by regulating zinc metabolism in the nervous system have not yet been attempted. There is a need to develop drugs capable of inhibiting brain damage through a mechanism that enhances lysosomal function by regulating intracellular zinc concentration and inhibiting PDE. Prior art literature

[0015] Published Patent Application No. 10-2022-0018552 The problem to be solved

[0016] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of brain injury comprising amlexanox.

[0017] The present invention also aims to provide a food composition for the prevention or improvement of brain damage comprising amlexanox.

[0018] The present invention also aims to provide a health functional food for the prevention or improvement of brain damage comprising amlexanox.

[0019] The present invention also aims to provide a composition for inducing an increase in the concentration of zinc within a lysosome comprising amlexanox.

[0020] The purposes of the present disclosure are not limited to those mentioned above, and other purposes and advantages of the present disclosure not mentioned may be understood from the following description and will be more clearly understood from the embodiments of the present disclosure. Furthermore, it will be readily apparent that the purposes and advantages of the present disclosure can be realized by the means and combinations thereof set forth in the claims. means of solving the problem

[0021] The present invention provides a pharmaceutical composition for the prevention or treatment of brain injury comprising amlexanox.

[0022] The chemical formula of the above-mentioned amlexanox is 2-amino-7-isopropyl-5-oxo-5H-[1]benzopyrano[2,3-b] pyridine-3-carboxylic acid, and the molecular formula is C 16 H 14 It is N2O4, and its molecular weight is 298.30. Amlexanox is odorless and has a yellowish-white crystalline powder, and its structural formula is as follows.

[0023] [Structural Formula 1]

[0024]

[0025] The aforementioned Amlexanox inhibits phosphodiesterase.

[0026] The above Amlexanox increases the amount of zinc in the lysosome.

[0027] The above-mentioned Amlexanox inhibits apoptosis caused by reactive oxygen species.

[0028] The above-mentioned Amlexanox inhibits the activity of astrocytes.

[0029] The above-mentioned Amlexanox is included at a concentration of 0.01 μM or more and less than 10.0 μM relative to the total amount of the composition.

[0030] The above brain damage is caused by a neurological disorder.

[0031] The present invention also provides a food composition for the prevention or improvement of brain damage comprising amlexanox.

[0032] The aforementioned Amlexanox inhibits phosphodiesterase.

[0033] The present invention also provides a health functional food for the prevention or improvement of brain damage comprising amlexanox.

[0034] The present invention also provides a composition for inducing an increase in the concentration of zinc in lysosomes, comprising amlexanox.

[0035] The aforementioned Amlexanox inhibits phosphodiesterase.

[0036] The above-mentioned Amlexanox induces acidification of the lysosome. Effects of the invention

[0038] The present invention may provide a pharmaceutical composition for the prevention or treatment of brain damage comprising amlexanox.

[0039] The present invention may also provide a food composition for the prevention or improvement of brain damage comprising amlexanox.

[0040] The present invention may also provide a health functional food for the prevention or improvement of brain damage comprising amlexanox.

[0041] The present invention may also provide a composition for inducing an increase in the concentration of zinc within a lysosome comprising amlexanox. Brief explanation of the drawing

[0042] Figure 1 shows the physiological mechanism of lysosome dysfunction and zinc in neurodegenerative brain diseases. Figure 2 shows the results of the phosphodiesterase inhibition experiment of Amlexanox. Figure 3 shows the results of measuring changes in intracellular zinc concentration due to amlexanox treatment. Figure 4 shows the change in pH and cathepsin activity of lysosomes due to amlexanox treatment. Figure 5 shows the effect of inhibiting cell death by amlexanox treatment. Figure 6 shows the results of confirming the effects of Amlexanox on neurological disorders and nerve damage caused by traumatic brain injury. Figure 7 shows the results of confirming whether motor nerves in a mouse model were recovered by Amlexanox treatment. Figure 8 shows the results of observing whether the activity of microglia was inhibited after brain injury by amlexanox treatment. Specific details for implementing the invention

[0043] The term "treatment" as used in the present invention refers to an approach to obtain beneficial or desirable clinical results, and includes alleviation of symptoms, reduction of the severity of the disease, a stabilized state of the disease (i.e., not worsening), delay or reduction in the rate of disease progression, improvement or temporary relief and alleviation of the disease state, regardless of whether beneficial or desirable clinical results are detectable or not for the purposes of the present invention, and whether partial or complete. The term "prevention" as used in the present invention includes, but is not limited to, any act of suppressing or delaying the disease using the above pharmaceutical composition. Accordingly, the present invention refers to both therapeutic treatments and preventive measures.

[0044] The pharmaceutical composition of the present invention may be administered at a therapeutically effective dose, which is the amount of the active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human as conceived by a researcher, veterinarian, physician, or other clinician, i.e., the amount that induces the alleviation of symptoms of the disease or disorder being treated. It is obvious to a person skilled in the art that the therapeutically effective dose and frequency of administration of the pharmaceutical composition of the present invention will vary according to the desired effect. Therefore, the optimal dose to be administered can be easily determined by a person skilled in the art and may be adjusted according to various factors including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health condition, gender and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and concurrently used drugs.

[0045] In the present invention, the term "food composition" refers to a food that acts favorably on one or more functions of an organism to provide a better state of health, regardless of the nutrients provided to the subject consuming it. Consequently, said food composition may be used for the prevention, improvement, or treatment of diseases or disease-causing factors.

[0046] The food composition of the present invention may preferably be formulated as a food composition by additionally including one or more carriers that are food-gradely acceptable or pharmaceutically acceptable in addition to the active ingredients described above.

[0047] In the present invention, "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body pursuant to Article 6727 of the Act on Health Functional Foods, and means a food with high medical and therapeutic effects that is processed to efficiently exhibit bio-regulatory functions such as prevention of brain damage, biological defense, immunity, and recovery for the purposes of the present invention, in addition to nutritional supply.

[0048] Of course, the amount of amlexanox mixed can be appropriately changed depending on the purpose of use (prevention, health, or therapeutic treatment), and it is preferable that the amlexanox be included in an amount of 0.01 to 95% by weight relative to the total weight of the food composition, and more preferably in an amount of 1 to 80% by weight. If the content is less than 0.01% by weight, the efficiency of administration may decrease, and if it exceeds 95% by weight, there may be difficulties in formulation.

[0049] There are no special restrictions on the types of food mentioned above. Examples of food to which the wood ear mushroom extract of the present invention can be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and include all health foods in the conventional sense.

[0050] When the amlexanox composition of the present invention is used as a health functional food, it may additionally include additional ingredients that do not have a harmful effect on the brain damage improvement or prevention effect of amlexanox.

[0052] The present invention will be further described below through examples, but it is obvious that the present invention is not limited by the following examples.

[0054] Test example

[0055] 1. Confirmation of Amlexanox's phosphodiesterase inhibitory effect

[0056] The relative activity (% activity) of Amlexanox (10 µM) was determined for various phosphodiesterase isomers, and experiments on the inhibition of phosphodiesterase by Amlexanox were conducted, with the results shown in Figure 2. As shown in Figure 2, it was confirmed that Amlexanox has an excellent inhibitory effect on all isoforms, including PDE3. In addition, to directly confirm the phenomenon of increased intracellular cAMP due to PDE inhibition, astrocytes from mice 3 days old were cultured, and Amlexanox was applied to the cells at approximately 2 weeks. After treating astrocytes with AMX and measuring changes in intracellular cAMP, it was confirmed that the cAMP level increased significantly. In addition, AD and ALS in vitro systems were constructed by injecting intracellular soluble amyloid beta and over-injecting Tau and SOD1 toxic proteins, and changes in toxic proteins induced by Amlexanox were confirmed. As a result, a decrease in toxic proteins was observed in all cell disease models.

[0057] 2. Confirmation of increase in intracellular zinc concentration induced by Amlexanox treatment

[0058] Figure 3 shows the results of measuring changes in intracellular zinc concentration after treatment with Amlexanox. Mouse astrocytes on the third day after birth were cultured on PLL-coated glass for two weeks, then stained for 30 minutes with zinc-staining samples FluoZin-3AM and Zinpyr-1 and lysosome-staining sample Lysotracter, and treated with Amlexanox. Consequently, changes in intracellular zinc, particularly within lysosomes, were confirmed using confocal microscopy. As a result, it was observed that the increase in zinc was located within the lysosomes.

[0060] 3. Confirmation of pH changes in lysosomes induced by Amlexanox treatment

[0061] Mouse astrocytes on the third day after birth were cultured on PLL-coated glass for two weeks. After staining with Lysosensor DND189, which can detect changes in lysosome pH, and Cathepsin L, known as an intralyseminal protein, the changes were observed by treating with AMX, and the results are shown in Figure 4. When Bafilomycin A1 is treated, the pH of the lysosome increases and the activity of lysosome-specific proteins decreases; however, when AMX is treated, it was observed that the pH becomes acidic while the activity of lysosome-specific proteins increases.

[0063] 4. Confirmation of the inhibitory effect of Amlexanox treatment on cell death

[0064] Mouse astrocytes on the third day after birth were cultured for two weeks, and then apoptosis was induced by treating with H2O2. The inhibitory effect of amlexanox was observed using an LDH assay, and the results are shown in Figure 5. It was confirmed that amlexanox inhibits apoptosis caused by reactive oxygen species.

[0066] 5. Confirmation of improvement in neurological disorders caused by traumatic brain injury by Amlexanox treatment

[0067] As shown in Figures 6 and 7, to investigate the effects of Amlexanox on neurological impairment and nerve damage caused by traumatic brain injury, one of the animal models of vascular dementia, Amlexanox (5 mg / kg) was administered once daily for one week after injury induction (Figure 6 B). To examine neurological impairment occurring after injury, the neurological severity score (NSS) test was performed and observed. The NSS is a score of 18 points measured by an investigator not involved in the treatment, evaluating reflexes, flexibility, motor skills, and coordination. A score of 1 indicates an inability to perform a specific task; higher scores indicate more severe impairment, while scores approaching 0 indicate good health. As a result, it was confirmed that the NSS score was lower in the group administered Amlexanox compared to the group administered a vehicle after traumatic brain injury (Figures 6 C and D). In addition, one week after traumatic brain injury, the activity of astrocytes and the number of neurons in both CA1 of the hippocampus and GCL and Hilus of the DG were significantly reduced, and it was found that this reduction in astrocyte activity and neurons was inhibited in the experimental group administered a novel PDE inhibitor (Fig. 6E). Furthermore, it was confirmed that motor neurons were recovered in a mouse model administered amlexanox (Fig. 7). This indicates that the administration of amlexanox improves neurological disorders occurring after traumatic brain injury and also inhibits astrocyte activity and neuronal death.

[0069] 6. Confirmation of Inhibition of Microglia Activation After Brain Injury by Amlexanox Treatment

[0070] We observed whether the activity of microglia after brain injury was inhibited by amlexanox treatment, and the results are shown in Figure 8. As shown in Figure 8, it was observed that amlexanox treatment inhibited the activity of microglia occurring after traumatic brain injury and significantly inhibited the activity of M1 microglia, which have an inflammatory phenotype.

[0072] From the experimental results above, it is indicated that Amlexanox controls glial cell activity and possesses neuroprotective effects, and furthermore, it is judged to have the potential to overcome neurological damage and cognitive impairment caused by vascular dementia.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 A composition for inducing an increase in the concentration of zinc in lysosomes and inhibition of M1 microglia activity reduced by traumatic brain injury in vitro, comprising amlexanox, wherein the composition inhibits the activity of M1 microglia, and the amlexanox is included at a concentration of 0.01 μM or more and less than 10.0 μM relative to the total amount of the composition. Claim 12 In claim 11, the composition is one that induces acidification of the lysosome, a composition for inducing an increase in the concentration of zinc in the lysosome reduced by traumatic brain injury in vitro and inhibiting the activity of M1 microglia. Claim 13 A method for increasing the concentration of zinc in lysosomes reduced by traumatic brain injury in vitro, comprising the step of administering to isolated cells a composition containing amlexanox for inducing an increase in the concentration of zinc in lysosomes reduced by traumatic brain injury in vitro and inhibiting the activity of M1 microglia, wherein the amlexanox is included at a concentration of 0.01 μM or more and less than 10.0 μM relative to the total amount of the composition, and the composition inhibits the activity of M1 microglia.

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